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J. Agric. Food Chem. 1999, 47, 2502−2504
RAPID COMMUNICATIONS Vitamins and Especially Flavonoids in Common Beverages Are Powerful in Vitro Antioxidants Which Enrich Lower Density Lipoproteins and Increase Their Oxidative Resistance after ex Vivo Spiking in Human Plasma Keywords: Lipoprotein oxidation; antioxidant; polyphenols; beverages INTRODUCTION
Consumption of fruits and vegetables is associated with a lowered risk of cardiovascular disease (Ames et al., 1993). One possible reason for this benefit is the presence of antioxidant vitamins C and E and the provitamin β-carotene in these foods. However, recent supplementation studies with vitamin E and β-carotene have cast doubt on this hypothesis (Rapola et al., 1997). Oxidation of low (LDL) and very low (VLDL) density lipoproteins are crucial steps in atherosclerotic lesion formation (Steinberg et al., 1989). Other dietary components, polyphenolic antioxidants such as flavonoids, have recently been shown in a seven-country epidemiology study to be protective for heart disease (Hertog et al., 1995). Beverages such as red wines and teas are high in polyphenols and provide a large quantity of flavonoids to the Mediterranean and Japanese diets, respectively. The increased consumption of red wines by the French has been used to explain the French Paradox of heart disease (Renaud and de Lorgeril, 1992). Tea drinking was found to reduce the risk of heart disease in a cross-cultural study (Hertog et al., 1995). We have recently shown that many polyphenols are stronger antioxidants than the vitamin antioxidants using the LDL + VLDL oxidation model (Vinson et al., 1995a). Polyphenols have also been found to bind to these lipoproteins (Vinson and Hontz, 1995) in plasma and protect them from ex vivo oxidation. This lipoprotein-bound antioxidant activity, i.e., enrichment, as measured by our methodology, has been shown to significantly correlate with protein binding (Wang and Goodman, 1999). By binding to lipoproteins, polyphenols can thus provide protection when the lipoproteins penetrate the endothelium of the aorta where they are subsequently oxidized (Steinberg et al., 1989). In this work, we have investigated the quality of the vitamin antioxidants and polyphenolic antioxidants in beverages and their ability to enrich lipoproteins. MATERIALS AND METHODS Pure compounds were obtained from Sigma or Aldrich Chemical Co. Beverages were obtained from local supermarkets. Teas and coffees were made fresh from leaves (Wang et al., 1994) and ground coffee (Fried et al., 1992), respectively. LDL + VLDL was isolated from a pool of normolipemic human plasma by affinity column (Vinson and Hontz, 1995). Wine was dealcoholized by low-temperature vacuum distillation. Phenol concentrations in the beverages were measured as catechin equivalents by the Folin-Cocialteu reagent. Beverages con-
taining ascorbic acid were treated with ascorbate oxidase before phenol measurement to remove ascorbate interference with the Folin reaction. The procedure for determination of the antioxidant concentration to inhibit in vitro LDL + VLDL oxidation 50% (IC50) has been previously published (Vinson et al., 1995a). Ex vivo plasma spiking of pure antioxidants, isolation of the lowdensity lipoproteins, and measurement of the lag time has been used to compare the effectiveness of lipoprotein-bound antioxidants (Vinson and Hontz, 1995). This is done by measuring the concentration to inhibit the isolated LDL + VLDL oxidation and thus increase the lag time by 50% (CLT50). In this study we also spiked pure antioxidants dissolved in water or methanol or diluted beverages (1-50 µL) at 50-200 µM (catechin equivalents) in the plasma (1 mL). Ubiquinol10 was formed from coenzyme Q10 (ubiquinone) by reduction with sodium dithionite. RESULTS AND DISCUSSION
The results are shown in Table 1 for pure antioxidant vitamins and nutrients, polyphenols, and beverages. Polyphenols generally were better antioxidants, as measured by IC50, than the vitamins. Among the vitamins and nutrients, ubiquinol-10 was the best antioxidant as was also the case with the LDL model oxidized with the peroxy radical generator AAPH (Thomas et al., 1997). Teas were found to contain high-quality antioxidants, which is not suprising since epigallocatechin gallate was the most powerful pure antioxidant with an IC50 of 0.08 µM (Vinson et al., 1995b) and it is the major polyphenol in green and black tea (Wang et al., 1994). The order of antioxidant quality for the beverages was black tea > coffee > prune juice ) beer > green tea > orange juice > red wine > tangerine juice > red grape juice > white grape juice > grapefruit juice. Ascorbic acid did not exhibit any lipoprotein-bound antioxidant activity. This is not surprising since it is very water soluble and has not been found to enrich lipoproteins after human consumption (Reaven et al., 1993). Tocopherol and retinol, two vitamin phenols, were active in our model and have been reported to bind to LDL in vitro and in vivo after supplementation (Reaven et al., 1993; Livrea et al., 1995). Ubiquinol-10 had good antioxidant properties after enrichment. It has been reported to bind to LDL and to be the first antioxidant in LDL to be oxidized (Stocker et al., 1991). As a result of these antioxidant properties and after absorption of CoQ10, ubiquinol-10 is also an in vivo phenolic antioxidant for LDL oxidation (Mohr et al., 1992). β-Carotene was a very poor in vitro antioxidant but did bind with the low-density lipoproteins LDL +
10.1021/jf9902393 CCC: $18.00 © 1999 American Chemical Society Published on Web 07/02/1999
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J. Agric. Food Chem., Vol. 47, No. 7, 1999 2503
Table 1. Comparison of Phenol Antioxidant Quality (IC50) and Lipoprotein-Bound Antioxidant Effectiveness (CLT50) of Vitamins, Polyphenols, and Beveragesa compound or beverage ascorbic acid tocopherol retinol β carotene ubiquinol-10 epicatechin (tea) catechin epigallocatechin gallate (tea) taxifolin (fruit juices) quercetin (fruit juices) quercetin rutinoside (citrus juice) hesperetin (citrus juice) hesperetin rutinoside (citrus juice) cyanidin (red wine, grape juice) resveratrol (red wine) chlorogenic acid (coffee) BEVERAGE orange juice tangerine juice grapefruit juice prune juice coffee black tea green tea white grape juice red grape juice Bordeaux red wine lager beer a
phenol content ×10-3(µM)
IC50 (µM)
CLT50 (µM)
1.45a 2.40a 1.90 4.30a 1.33 0.19a
0.21a 0.33a 0.30a
inactive 54b 103 134 65 72b 85 42b 38 59b 108b inactive inactive 120b 59b 108b
0.42 0.50 0.95 0.30 0.26 0.23 0.38 0.70 0.65 0.45 0.30
inactive inactive inactive 111 106 67 66 93 30 27 390
0.08a 0.34 0.22a 0.51a 3.66
0.77 1.38 1.59 5.63 10.32 17.40 12.00 2.88 7.66 9.58 10.13
been found to have no antioxidant activity (BenaventeGarcı´a et al., 1997). The methoxyflavanone hesperetin did not have any lipoprotein-bound antioxidant activity. The best beverages were by far the red wine and red grape juice. These were followed by the two teas (green and black were equal) with beer being the poorest antioxidant in this model. The beverages, however, were quite good compared with the individual polyphenols present in the beverages. For instance, red wine was much better than cyanidin, which is an anthocyanin. Anthocyanins are the major class of antioxidant polyphenols present in red wine (Ghiselli et al., 1998). Thus, there may additive or synergistic effects of the polyphenols in the beverages when they bind to the lipoproteins which will be tested in future experiments. The lipoprotein-bound antioxidant activity of the teas and wines shows that ex vivo plasma spiking can mimic the in vivo situation since drinking red wine (Fuhrman et al., 1995) and teas (Ishikawa et al., 1997) has been shown to produce in vivo LDL enrichment and a decreased LDL oxidizability. Conversely, orange juice, which had no lipoprotein-bound antioxidant activity in our model, had no in vivo lipoprotein antioxidant activity after human supplementation (Abbey et al., 1995). This ex vivo model is of course limited as the polyphenols in beverges are metabolized after absorption in vivo and therefore chemically changed. It remains to be shown whether the other active beverages in our model such as grape juice, prune juice, coffee, and beer have in vivo antioxidant activity.
Vinson et al., 1995a. b Vinson et al., 1995b.
LITERATURE CITED
Figure 1. Ex vivo spiking of diluted beverages in plasma at 100 µM and the effect on the cupric ion oxidation of isolated LDL + VLDL as measured by conjugated diene formation at 234 nm.
VLDL as found previously for LDL (Romanchik et al., 1997). Although it is very lipophilic and binds to LDL, it was not very effective in decreasing the oxidative susceptibility of LDL isolated after ex vivo spiking. The order of polyphenol effectiveness of lipoproteinbound antioxidant activity is epigallocatechin gallate ≈ taxifolin > quercetin ≈ resveratrol . quercetin rutinoside ≈ chlorogenic acid > cyanidin. Hesperetin and hesperetin rutinoside (hesperidin) did not exhibit any lipoprotein-bound antioxidant activity. Representative lipoprotein oxidation curves after beverage spiking into plasma are shown in Figure 1. Among the beverages, the citrus juices such as orange, tangerine, and grapefruit did not have any antioxidant effect for the lipoproteins isolated after plasma spiking. This is not surprising since some major polyphenols in citrus juices are methoxyflavanones, some of which have
Abbey, M.; Noakes, M.; Nestel, P. J. Dietary supplementation with orange juice and carrot juice in cigarette smokers lower oxidation products in copper-oxidized low-density lipoproteins. J. Am. Diet. Assoc. 1995, 95, 671-675. Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. 1993, 90, 7915-7922. Benavente-Garcı´a, O.; Castillo, J.; Marin, F. R.; Ortun˜o, A.; Del Rı´o, J. A. Use and Properties of citrus bioflavonoids. J. Agric. Food Chem. 1997, 45, 4505-4515. Fried, R. E.; Levine, D. M.; Kwiterovich, P. O.; Diamond, E. L.; Wilder, L. B.; Moyh, R. F.; Pearson, T. A. The effect of filtered coffee consumption on plasma lipids. J. Am. Med. Assoc. 1992, 267, 811-815. Fuhrman, L. A.; Aviram, M. Consumption of red wine with meals reduces the susceptibility of human plasma and lowdensity lipoprotein to lipid oxidation. Am. J. Clin. Nutr. 1995, 61, 549-554. Ghiselli, A.; Nardini, M.; Baldi, A.; Scaccini, C. Antioxidant activity of different phenolic fractions separated from an Italian red wine. J. Agric. Food Chem. 1998, 46, 361-367. Hertog, M. G. L.; Kromhout, D.; Aravanis, C.; Blackburn, H.; Buzina, R.; Fidanza, F.; Giampaoli, S.; Jansen, A.; Menotti, A.; Nedeljkovic, S.; Pekkarinen, M.; Simic, B. S.; Toshima, H.; Feskens, E. J. M.; Hollman, P. C. H.; Katan, M. B. Flavonoid intake and long-term risk of coronary heart disease and cancer in the seven countries study. Arch. Intern. Med. 1995, 155, 381-386. Ishikawa, T.; Suzukawa, M.; Ito, T.; Yoshida, H.; Ayaori, M.; Nishikawa, M.; Yonemura, A.; Hara, Y.; Nakamura, H. Effect of tea flavonoid supplementation on the susceptibility of low-density lipoprotein oxidative modification. Am. J. Clin. Nutr. 1997, 66, 261-266. Jialal, I. C.; Fuller, C. J.; Huet, B. A. The effect of alphatocopherol supplementation on LDL oxidation: a doseresponse study. Atheroscler. Thromb. Vasc. Biol. 1995, 15, 190-198.
2504 J. Agric. Food Chem., Vol. 47, No. 7, 1999 Livrea, M. A.; Tesoriere, L.; Riccio, A. Contribution of vitamin A to the oxidative resistance of human low density lipoprotein. Free Rad. Biol. Med. 1995, 18, 401-409. Mohr, D.; Bowry, V. W.; Stocker, R. Dietary supplementation with coenzyme Q10 results in increased levels of ubiquinol10 within circulating lipoproteins and increased resistance of human low-density lipoprotein to the initiation of lipid peroxidation. Biochim. Biophys. Acta 1992, 1126, 247-254. Rapola, J. M.; Virtamo, J.; Ripatti, S.; Huttunen, J. K.; Albanes, D.; Taylor, P. R.; Heinonen, O. P. Randomised trial of alpha-tocopherol and β-carotene supplements on incidence of major coronary events in men with previous myocardial infarction. Lancet 1997, 349, 1715-1720. Reaven, P. D.; Khouw, A.; Beltz, W. F.; Partharasarthy, S.; Witzum, J. L. Effect of dietary antioxidant combinations in humans. Protection of LDL by vitamin E but not by β-carotene. Arterioscler. Thromb. 1993, 13, 590-600. Renaud, S.; de Lorgeril, M. Wine, alcohol, platelets, and the French paradox for coronary heart disease. Lancet 1992, 339, 1523-1526. Romanchik, J. E.; Harrison, E. H.; Morel, D. W. Addition of lutein, lycopene, or β-carotene to LDL or serum in vitro: effects on carotenoid distribution, LDL composition and LDL oxidation. Nutr. Biochem. 1997, 8, 681-688. Steinberg, D.; Parathasarathy, S.; Carew, T. E.; Khoo, J. C.; Witzum, J. L. Beyond cholesterol; modification of lowdensity lipoprotein that increases its atherogenecity. New Engl. J. Med. 1989, 320, 915. Stocker, R., Bowry, V. W.; Frei, B. Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxidation than does R-tocopherol. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1646-1650. Thomas, S. R.; Neuzil, J.; Stocker, R. Inhibition of LDL oxidation by ubiquinol-10. A protective mechanism for coenzyme Q10 in atherogenesis? Mol. Aspects Med. 1997, 18, s85-s103. Vinson, J. A.; Hontz, B. A. Phenol antioxidant index: comparative antioxidant effectiveness of red and white wines. J. Agric. Food Chem. 1995, 43, 401-403.
Rapid Communications Vinson, J. A.; Dabbagh, Y. A.; Serry, M. M.; Jang, J. Plant flavonoids, especially tea flavonols, are powerful antioxidants using an in vitro oxidation model for heart disease. J. Agric. Food Chem. 1995a, 43, 2800-2802. Vinson, J. A.; Jang, J.; Dabbagh, Y. A.; Serry, M. M.; Cai, S. Plant polyphenols exhibit lipoprotein-bound antioxidant activity using an in vitro model for heart disease. J. Agric. Food Chem. 1995b, 43, 2798-2799. Wang, W.; Goodman, M. T. Antioxidant property of dietary phenolic agents in a human LDL-oxidation ex vivo model: interaction of protein binding activity. Nutr. Res. 1999, 19, 191-202. Wang, Z. Y.; Huang, M. T.; Lou, Y. R.; Xie, J. G.; Reuhl, K. R.; Newmark, H. I.; Ho, C. T.; Yang, C. S.; Conney, A. H. Inhibitory effects of black tea, green tea, decaffeinated black tea and decaffeinated green tea on ultraviolet β light-induced skin carcinogenesis in 7-12-dimethylbenz[R]anthraceneinitiated SKH-1 mice. Cancer Res. 1994, 54, 3428-3435.
Joe A. Vinson,* Jinhee Jang, Jihong Yang, Yousef Dabbagh, Xiquan Liang, Mamdouh Serry, John Proch, and Songhuai Cai Department of Chemistry, The University of Scranton, Scranton, Pennsylvania 18510-4626 Received for review March 8, 1999. Revised manuscript received June 4, 1999. Accepted June 5, 1999. The authors gratefully acknowledge financial support by Welch Foods, Inc., Thomas J. Lipton Co., and the Florida Citrus Commission. JF9902393 * To whom correspondence should be addressed. (telephone (570) 941-7551; fax (570) 941-7510; e-mail
[email protected]).
